Ultrasensitive, Multiplexed Buoyant Sensor for Monitoring Cytokines in Biofluids

Multiplexed quantification of low-abundance protein biomarkers in complex biofluids is important for biomedical research and clinical diagnostics. However, in situ sampling without perturbing biological systems remains challenging. In this work, we report a buoyant biosensor that enables in situ monitoring of protein analytes at attomolar concentrations with a 15 min temporal resolution. The buoyant biosensor implemented with fluorescent nanolabels enabled the ultrasensitive and multiplexed detection and quantification of cytokines. Implementing the biosensor in a digital manner (i.e., counting the individual nanolabels) further improves the low detection limit. We demonstrate that the biosensor enables the detection and quantification of the time-varying concentrations of cytokines (e.g., IL-6 and TNF-α) in macrophage culture media without perturbing the live cells. The easy-to-apply biosensor with attomolar sensitivity and multiplexing capability can enable an in situ analysis of protein biomarkers in various biofluids and tissues to aid in understanding biological processes and diagnosing and treating diverse diseases.

format with a sampling duration from 15 minutes to 2 hours at 37°C.Subsequently, the buoyant sensors were transferred into a 96-well microplate and rinsed with wash buffer (0.05% Tween-20 in 1X PBS) 3 times.The sensors were then incubated with the biotinylated detection antibody (50 ng/mL in reagent diluent for both IL-6 and TNF-α) for 2 hours, followed by the wash buffer rinsing 3 times, 100 µL PF nanolabel solution (optical density ~0.5) was added and incubated for 30 minutes, followed by rinsing 3 times.Finally, the sensors were imaged using LI-COR fluorescence imager and BioTek Cytation 5 Cell Imaging Multi-Mode Reader.For imaging analysis, at least three microdots were used to obtain mean fluorescent intensity and nanolabel numbers and corresponding standard deviations.To monitor IL-6 and TNF-α in macrophage culture, the sensors were introduced to the surface of culture media at different times and floated for 15 minutes to capture the targets.All other steps follow the same for IL-6 and TNF-α quantification.To validate the sensor accuracy, we performed spike-andrecovery and linearity-of-dilution experiments.In spike-and-recovery experiments, 90 µL macrophage culture supernatant was spiked with 10 µL pristine cell culture medium (RPMI-1640 with 10% FBS and 1% pen-strep) containing 0, 250 pg/mL, 500 pg/mL, and 1 ng/mL IL-6.Standard IL-6 in pristine culture medium at concentrations of 0, 25 pg/mL, 50 pg/mL, and 100 pg/mL served as controls.The IL-6 concentrations in the controls and samples were quantified using our sensors, and percentage recovery was calculated using the equation: Recovery rate = [(observed sample spike concentration -observed sample initial concentration) / expected concentration] x 100% (Table S1).In linearity-of-dilution experiments, macrophage culture supernatant was diluted with pristine culture medium at different dilution factors of 1:2, 1:4, and 1:8 and then quantified with our sensors.Percentage recovery was calculated using the equation: Recovery rate = (observed sample concentration / expected concentration) x 100% (Table S2).
Macrophage culture.The THP-1 cell culture follows the previous protocol 5 .RPMI-1640 media supplemented with 10% FBS and 1% pen-strep was used to culture THP-1 cells.For differentiation into macrophages, the THP-1 cells cultured in the 24-well microplate with a cell density of 200,000 cells/cm 2 were treated with the culture medium containing 50 ng/mL PMA.It took 24 hours for the THP-1 cells to differentiate and adhere to the microplate.The fresh cell culture media (RPMI 1640 medium supplemented with 10% FBS and 1% pen-strep) with 100 ng/mL LPS and 15 ng/mL IFN-γ were added to classically activate M0 into M1.For live/dead viability staining, the cells were rinsed twice with sterile Dulbecco's PBS (DPBS) to thoroughly remove the culture medium and then immersed in the mixture of Calcein AM (2 μM in DPBS) and Ethidium Homodimer-1 (4 μM in DPBS) for 30 minutes at room temperature.The cells were imaged under a fluorescence microscope (Zeiss Axio Observer 3) with red (ex/em 495 nm/635 nm) and green (ex/em 495 nm/515 nm) channels to observe dead and live cells, respectively.

Figure S1 .
Figure S1.PS film cut with different methods.(a) Optical images of PS film cut by CO2 laser laminated on PDMS surface.The edge of circular PS film shows plastic deformation and materials degradation.(b) Optical image of PS cut by picosecond-fast laser pulses laminated on PDMS surface.(c) Fluorescence image of a buoyant sensor after pFLISA showing unwanted fluorescence background at the edge of the sensor, resulting from the defects introduced during PS cutting shown in (a).

Figure S3 .
Figure S3.(a) Fluorescence intensity images of the sensors exposed to 100 fg/mL of IL-6 and blank (reagent dilute without IL-6) following pFLISA.(b) Zoomed-in IL-6 dose-dependent fluorescence intensity at a low concentration range.

Figure S5 .
Figure S5.Percent mean relative error and coefficient of variation in back-calculated concentrations at the concentration range from 0.1 pg/mL to 100 pg/mL.

Figure S6 .
Figure S6.IL-6 dose-dependent fluorescence intensity on the buoyant sensors following pFLISA with 30-min sampling time.

Figure S7 .
Figure S7.(a) Enlarged Fluorescence microscopy images and (b) enlarged Gaussian blur processed images of the buoyant sensors exposed to different IL-6 concentrations shown in Figure 3d-3e.

Figure S8 .
Figure S8.(a) Fluorescence microscopy images and (b) Gaussian blur processed images of the buoyant sensors exposed to different IL-6 concentrations used for digital analysis.

Figure S9 .
Figure S9.IL-6 dose-dependent fluorescence digital counting for an extended range of IL-6 concentrations following pFLISA with 15-min sampling time.

Figure S10 .
Figure S10.IL-6 dose-dependent fluorescence digital counting following pFLISA with 15-min sampling time.A linear fitting was applied after removing the blank data point.

Figure S11 .
Figure S11.(a) Fluorescence intensities from mouse IL-6 capture antibodies after exposure to different concentrations of mouse and human IL-6 and TNF-α.(b) Fluorescence intensities from mouse TNF-α capture antibodies after exposure to different concentrations of mouse and human TNF-α and IL-6.

Figure S12 .
Figure S12.Fluorescence intensities resulting from IL-6 at different concentrations measured from the freshly prepared sensors and the glycerol-coated sensors after storage at 4 °C for 1 day and 3 days.

Figure S13 .
Figure S13.Overlaid fluorescence images of M0 and M1 macrophages without exposure to buoyant sensors after live/dead cell staining.

Figure S14 .
Figure S14.Overlaid fluorescence images of cells cultured under standard conditions (control) and with exposure to sensor for 3 days after live/dead cell staining.

Figure S15 .
Figure S15.TNF-α dose-dependent fluorescence intensity on the buoyant sensors with pristine and Gamma irradiated PS following pFLISA.

Table S1 .
Spike and recovery of IL-6 in macrophage culture supernatant.

Table S2 .
Linearity-of-dilution results for IL-6 in macrophage culture supernatant.